Pearson Edexcel International A Level Biology: Culturing Microorganisms and Bacterial Growth (YBI11)

This guide teaches culturing microorganisms and bacterial growth from scratch for the Pearson Edexcel International Advanced Subsidiary/Advanced Level Biology specification (YBI11), Issue 2. It covers statements 6.1 to 6.4 of Topic 6, including Core Practical 13. Topics 1 to 4 are International AS (IAS) and Topics 5 to 8 are International A2 (IA2), so this material is A2. There are no tiers. Pair it with the revision notes and the practice questions. The course hub and the printable checklist show the rest of the course, and the free diagnostics show where to start. Conditions for bacterial growth and Core Practical 9 are in the Topic 4 guide.

What this guide covers

Statement	What you must be able to do
6.1	Understand the principles and techniques of culturing microorganisms, using aseptic technique
6.2	Understand methods of measuring growth: cell counts, dilution plating, mass and optical methods (turbidity)
6.3	Understand the phases of a bacterial growth curve (lag, exponential, stationary, death) and calculate exponential growth rate constants
6.4 (Core Practical 13)	Investigate the rate of growth of microorganisms in a liquid culture, taking into account the safe and ethical use of organisms

6.1 Culturing microorganisms with aseptic technique

To culture a microorganism is to provide conditions in which it grows and divides in a controlled way. Two aims run through every technique:

• Keep unwanted microorganisms out, so the culture is pure and your results mean something.
• Keep the culture in, so you and others are not exposed to it.

Culture media. A medium supplies a carbon source (often glucose), a nitrogen source, mineral ions, and sometimes growth factors such as vitamins. A liquid medium is a broth. A solid medium is made by adding agar, a gel from seaweed that most bacteria cannot digest, so it stays solid as the colonies grow. Media and equipment are sterilised first, usually in an autoclave, which uses steam under pressure at about 121 °C.

Aseptic technique is the set of habits that keep the work clean:

• Wipe the bench with disinfectant. Work near a lit Bunsen burner, whose rising warm air carries drifting microbes away from open vessels.
• Flame the inoculating loop until it glows red, then let it cool in the air before touching the culture, otherwise it kills the cells you want.
• Open lids only as far and for as long as needed. Pass the neck of a bottle through the flame after opening and before closing.
• Use sterile pipettes and spreaders and discard them for autoclaving.
• Seal an inoculated plate with short pieces of tape, not all round. Oxygen can still enter, which discourages growth of anaerobes that could be harmful. Incubate plates inverted so condensation does not drip onto the colonies.
• Incubate at a modest temperature, such as 25 °C, not body temperature, which would favour any human pathogen that got in. Use a safe laboratory strain only.
• Do not open plates after incubation. Sterilise cultures and equipment before disposal.

Making a pure culture on a plate. Streaking spreads a small sample thinly over agar so that single cells end up far apart. Each cell divides to form a visible colony. A colony is a clone of one cell.

6.2 Measuring the growth of microorganisms

Cell counts

A haemocytometer is a slide with a counting chamber of known depth and a ruled grid. A drop of culture is placed under a cover slip and cells over the grid are counted under a microscope. Cells touching two of the edges of a square are counted and those touching the other two ignored, so none is counted twice. Living and dead cells are both counted unless a stain tells them apart: methylene blue stains dead yeast cells blue, while living cells stay colourless. The method suits larger cells such as yeast.

The chamber depth is 0.1 mm. One large square is 1 mm × 1 mm, so its volume is 0.1 mm³, which is 1 × 10⁻⁴ cm³.

Worked example 1. A yeast culture is diluted 1 in 10. The mean count is 18 cells per large square. Find the concentration in the original culture.

• Cells per cm³ in the diluted sample = 18 ÷ (1 × 10⁻⁴) = 1.8 × 10⁵.
• Multiply by the dilution factor: 1.8 × 10⁵ × 10 = 1.8 × 10⁶ cells cm⁻³.

Dilution plating (viable counts)

A culture of bacteria is far too dense to count on a plate, so it is diluted in a series. In a serial dilution, 1 cm³ of culture is added to 9 cm³ of sterile diluent to give a 10⁻¹ dilution, and 1 cm³ of that to a further 9 cm³ to give 10⁻². This is repeated for the number of steps needed. A known volume of the chosen dilutions is spread on agar plates and incubated. Each colony is assumed to have grown from one viable cell, which is a cell able to divide. The result is given as colony-forming units (CFU) per cm³.

Choose plates with a countable number of colonies. A common target is about 30 to 300, because fewer than that is statistically unreliable and more than that merge. Plate in triplicate and calculate a mean.

CFU per cm³ of original = (mean colonies ÷ volume plated in cm³) × dilution factor

Worked example 2. Three plates of the 10⁻⁵ dilution, each given 0.1 cm³, show 43, 51 and 47 colonies.

• Mean = (43 + 51 + 47) ÷ 3 = 47.
• 47 ÷ 0.1 = 470 CFU cm⁻³ in the 10⁻⁵ tube.
• Multiply by 10⁵: 4.7 × 10⁷ CFU cm⁻³ in the original culture.

Dilution plating counts only living cells that can form colonies. Clumps of cells give one colony, so it can underestimate.

Mass

Cells in a known volume of culture are collected by centrifuging or filtering, washed, and dried in an oven until the mass stops changing. This dry mass is the mass of the cells. Dry mass is preferred to wet mass because wet mass includes a variable amount of water. It cannot tell living cells from dead.

Worked example 3. A 10 cm³ sample has a dry mass of 0.012 g at the start and 0.084 g later. Give the increase in g dm⁻³.

• Increase = 0.084 − 0.012 = 0.072 g in 10 cm³.
• 1 dm³ = 1000 cm³, so multiply by 100: 7.2 g dm⁻³.

Optical methods: turbidity

Bacteria scatter light, so a dense culture looks cloudy. A colorimeter (or spectrophotometer) measures how much light passes through a sample, shown as absorbance or percentage transmission. More cells scatter more light, so absorbance rises as the culture grows. It is quick and can be repeated on the same culture.

To use it well:

• Zero the instrument with a cuvette of sterile medium (a blank), and mix the culture before each reading.
• Build a calibration curve: measure the absorbance of samples whose cell concentration is known by another method, then read unknown concentrations from the graph.

Limits: it includes dead cells, and at high density absorbance is no longer proportional to cell number, so dense samples may need diluting.

6.3 The bacterial growth curve

Bacteria divide by binary fission, so a population doubles each generation. Plotted against time, the number of viable cells in a closed batch culture follows four phases.

Time (h)	Viable cells (cm⁻³)	log₁₀ of cells	Phase
0	1.0 × 10⁴	4.00	Lag
1	1.1 × 10⁴	4.04	Lag
2	2.5 × 10⁴	4.40	Exponential
4	8.9 × 10⁴	4.95	Exponential
6	3.2 × 10⁵	5.51	Exponential
8	6.2 × 10⁵	5.79	Slowing
10	6.8 × 10⁵	5.83	Stationary
12	6.8 × 10⁵	5.83	Stationary
16	2.0 × 10⁵	5.30	Death

• Lag phase. Cells adjust to the new medium, synthesising enzymes and taking up nutrients. Little division happens.
• Exponential (log) phase. Cells divide at a constant, maximum rate and numbers double at regular intervals. A graph of log₁₀ number against time is a straight line.
• Stationary phase. Nutrients run low and waste products, such as acids or alcohol, build up. The rate of cell division equals the rate of cell death, so the number of viable cells stays roughly constant.
• Death phase. The rate of cell death is greater than the rate of division, because nutrients are exhausted and waste is toxic, so viable cells fall.

A turbidity reading would not fall as far in the death phase, because dead cells still scatter light.

Exponential growth rate constants

Over a time t, a population of N₀ cells that undergoes n generations has N = N₀ × 2ⁿ cells. Taking logs gives

n = (log₁₀ N − log₁₀ N₀) ÷ log₁₀ 2 = (log₁₀ N − log₁₀ N₀) ÷ 0.301

The growth rate constant, k, is the number of generations per unit time, k = n ÷ t. The generation time, g, is the time for one doubling, g = 1 ÷ k. On a graph of log₁₀ N against t, the gradient in the exponential phase equals 0.301 × k.

Use only exponential-phase values for k. If a question gives its own formula for k, follow that.

Worked example 4. Use the table: 2.5 × 10⁴ cells cm⁻³ at 2 h and 3.2 × 10⁵ at 6 h.

• log₁₀ (3.2 × 10⁵) − log₁₀ (2.5 × 10⁴) = 5.505 − 4.398 = 1.107.
• n = 1.107 ÷ 0.301 = 3.68 generations.
• k = 3.68 ÷ 4 h = 0.92 generations per hour.
• g = 1 ÷ 0.92 = 1.09 h = 65 minutes.

Worked example 5. A culture starts exponential growth at 3.0 × 10³ cells cm⁻³ with a generation time of 45 minutes. Find the number after 6 hours.

• k = 60 ÷ 45 = 1.33 generations per hour.
• n = 1.33 × 6 = 8 generations (360 ÷ 45).
• N = 3.0 × 10³ × 2⁸ = 7.7 × 10⁵ cells cm⁻³.

6.4 Core Practical 13: growth in a liquid culture

The specification asks you to investigate the rate of growth of microorganisms in a liquid culture, taking into account the safe and ethical use of organisms.

A typical design. Inoculate flasks of sterile nutrient broth with equal volumes of a safe strain, such as yeast or a harmless laboratory bacterium, and incubate them under different values of one independent variable.

• Independent variable: one of temperature, glucose concentration, pH or initial inoculum.
• Dependent variable: growth, measured as absorbance in a colorimeter at set times (for example every 30 minutes), or as viable counts from dilution plating.
• Controlled variables: volume and type of broth, volume of inoculum, starting cell density, flask size, shaking or aeration, and measuring time.
• Control: a flask of sterile broth with no inoculum, which shows that the absorbance rise is due to growth and not contamination.

Analysis. Plot log₁₀ cell number or a suitable measure against time. Find the exponential phase from the straight section, then calculate the growth rate constant, or compare gradients between conditions. Make repeats and calculate means.

Safety. Complete a risk assessment first. Use only approved strains, keep cultures closed, incubate at a modest temperature, treat spills with disinfectant, sterilise all waste and wash your hands.

Ethics. Microorganisms raise fewer concerns than animals, but use them responsibly: never release cultures, never grow unknown organisms, and sterilise waste before disposal.

Evaluating. Turbidity includes dead cells, sampling may contaminate a flask, and incubator temperatures can vary.

Common errors

• Taking the colonies on a plate as the number in the original culture without the dilution factor.
• Mislabelling the dilution: the fifth tube of a 1 in 10 series is 10⁻⁵, not 10⁻⁴.
• Calling the stationary phase “no growth”, when division and death balance.
• Saying turbidity falls in the death phase just as viable count does.
• Calculating k with stationary or lag-phase values.
• Counting cells on all four edges of a haemocytometer square.

Now test yourself with the practice questions and compress the content with the revision notes.

Official syllabus

Pearson Edexcel International Advanced Subsidiary/Advanced Level Biology (YBI11) specification, Issue 2, published by Pearson Education Limited.